Engeletin attenuates the inflammatory response via inhibiting TLR4-NFκB signaling pathway in Crohn's disease-like colitis.

ETHNOPHARMACOLOGICAL RELEVANCE: Smilax glabra rhizome has a long history been used for clinical purposes in traditional Chinese medicinal for treating various inflammatory conditions. Engeletin1 (ENG) is one of the most abundant bioactive compounds found in Smilax glabra rhizome, with anti-inflammatory, antioxidant, and ulcer-preventing activities. AIM OF THE STUDY: The purpose of this study was to investigate the ability of ENG to alleviate inflammatory symptoms and improve epithelial barrier integrity utilize a 2,4,6-trinitrobenzene sulfonic acid2 (TNBS)-induced murine model in Crohn's disease3 (CD)-like colitis, and to characterize the underlying anti-inflammatory mechanisms of action. MATERIALS AND METHODS: A colitis model was established in BALB/c mice and treated with ENG for 7 days. RAW264.7 macrophages were pre-treated with ENG and lipopolysaccharide4 (LPS) stimulation. The mice's weight and colon length were assessed. qPCR and Western blotting were used to analyze gene expression and TLR4-NFκB pathway. Flow cytometry was used to analyze the polarization states of the macrophages. RESULTS: Treatment with ENG was sufficient to significantly alleviate symptoms of inflammation and colonic epithelial barrier integrity in treated mice. Significant inhibition of TNF-α, IL-1ß, and IL-6 expression was observed following ENG treatment in vivo and in vitro. ENG was also determined to be capable of inhibiting the expression of iNOS and CD86, inhibited M1 macrophage polarization in vitro, as well as the TLR4-NFκB signaling pathway. Molecular docking showed a highly stable binding between ENG and TLR4. CONCLUSION: ENG has been proven to alleviate inflammation and ameliorate the damage of epithelial barrier in CD-like colitis. ENG also suppressed the M1 macrophages polarization and the inhibited inflammatory cytokines. TLR4-NFκB signaling pathway, especially TLR4, may be the target of ENG. These data offer a new insight into the therapeutic mechanisms of ENG.

[Protective effect and mechanism of Zuogui Jiangtang Jieyu Formula on damage to hippocampal synaptic microenvironment in rats with diabetes-related depression based on microglia-neuron crosstalk signal CD300f/TLR4].

This study aims to reveal the protective effect and mechanism of Zuogui Jiangtang Jieyu Formula on the damage to hippo-campal synaptic microenvironment in rats with diabetes-related depression(DD) via regulating microglia immune receptor molecule-like family member f(CD300f)/Toll-like receptor 4(TLR4) signal. Firstly, the model of DD rats was established by a two-week high-fat diet+STZ injection+chronic mild and unpredictable stress plus isolation for 28 days. The rats were randomly divided into normal group, model group, CD300f blocker(CLM1, 2 µg·kg~(-1)) group, CD300f agonist(Fcγ, 5 µg·kg~(-1)) group, positive drug(0.18 g·kg~(-1) metformin+1.8 mg·kg~(-1) fluoxetine) group, and high-dose and low-dose(20.52 and 10.26 g·kg~(-1)) Zuogui Jiangtang Jieyu Formula groups. Depression-like behavior of rats was evaluated by open field and forced swimming experiments. The levels of blood glucose and insulin were detected by biochemical analysis. The levels of tumor necrosis factor α(TNF-α), interleukin-1ß(IL-1ß), indoleamine 2, 3-dioxygenase(IDO), 5-hydroxytryptamine(5-HT), and dopamine(DA) in the hippocampus were detected by enzyme-linked immunosorbent assay. The changes in the synaptic ultrastructure in hippocampal neurons of rats were observed by transmission electron microscopy. The protein expressions of CD300f, TLR4, synaptophysin(SYN), and postsynaptic density protein 95(PSD-95) in microglial cells of the hippocampus were detected by immunofluorescence and Western blot. The results indicated that compared with that in the normal group, the total movement distance in open field experiments was reduced in the model group, and the immobility time in forced swimming experiments increased, with an elevated insulin level in serum, as well as TNF-α, IL-1ß, and IDO levels in the hippocampus. The 5-HT and DA levels in the hippocampus were reduced. In addition, the CD300f expression was down-regulated in microglial cells of the hippocampus, and the TLR4 expression was up-regulated. Moreover, the expression of synapse-related proteins SYN and PSD-95 in hippocampal neurons decreased, and the synaptic ultrastructure of hippocampal neurons was significantly damaged. Compared with the model group, the CD300f blocker and agonist aggravated and alleviated the above abnormal changes, respectively. High-dose and low-dose Zuogui Jiangtang Jieyu Formula could significantly improve the above depression-like beha-vior in rats, inhibit the abnormal increase of TNF-α, IL-1ß, and IDO and the decrease of 5-HT and DA, effectively increase the expression of CD300f in microglial cells, and decrease the expression of TLR4. They could up-regulate the protein expression of presyna-ptic membrane SYN and postsynaptic membrane PSD-95 in hippocampal neurons and finally improve the damage to the hippocampal synaptic microenvironment. In conclusion, this research confirmed that Zuogui Jiangtang Jieyu Formula effectively alleviated the depression-like behavior and inhibited inflammatory activation of microglial cells in the hippocampus of rats with DD, and the mechanism might be related to the regulation of CD300f/TLR4 signal to alleviate the damage to hippocampal synaptic microenvironment.

[Effect of Polygonati Rhizoma aqueous extract on chronic obstructive pulmonary disease in rats].

This study aimed to investigate the effects and potential mechanism of Polygonati Rhizoma aqueous extract on chronic obstructive pulmonary disease(COPD) in rats. Forty-eight Sprague-Dawley rats were randomly assigned to the normal, model,Yupingfeng Granules(1. 5 g·kg~(-1)), and low-, medium-, and high-dose(0. 25, 0. 5, and 1 g·kg~(-1), respectively) Polygonati Rhizoma aqueous extract groups. The rat model of COPD was established by cigarette smoke inhalation for 8 weeks, and then the modeled rats received corresponding treatment for 4 weeks. The grip strength and fecal moisture content were measured, and the lung index was calculated. Enzyme-linked immunosorbent assay(ELISA) was employed to determine the levels of interleukin(IL)-6 and tumor necrosis factor(TNF)-α in the lung tissue. Hematoxylin-eosin(HE) staining and Masson staining were performed to assess the pathological changes in the lung tissue. Flow cytometry was used to analyze T lymphocytes and their subpopulations in the peripheral blood, and the immunofluorescence assay and Western blot were employed to measure the protein levels of Toll-like receptor 4(TLR4), phosphorylated nuclear factor-kappaB(p-NF-κB), NF-κB, phosphorylated inhibitory kappa B-α(p-IκBα), IκBα, IL-6,and TNF-α in the lung tissue. The results indicated that the treatment with Polygonati Rhizoma aqueous extract significantly reduced the fecal moisture content, enhanced the grip strength, and inhibited inflammatory infiltration and fibrosis in the lung tissue. The treatment increased the Th/Tc ratio and Th cell proportion and decreased the Tc cell proportion in the peripheral blood. Furthermore,the treatment down-regulated the expression levels of TLR4, IL-6, and TNF-α and the p-NF-κB/NF-κB and p-IκBα/IκBα ratios in the lung tissue. In conclusion, Polygonati Rhizoma aqueous extract can ameliorate lung tissue damage in the rat model of COPD by inhibiting the TLR4/NF-κB signaling pathway and the production of inflammatory mediators.

Activation of the NLRP1B inflammasome by caspase-8.

Cleavage of the innate immune receptor NLRP1B by various microbial proteases causes the proteasomal degradation of its N-terminal fragment and the subsequent release of a C-terminal fragment that forms an inflammasome. We reported previously that metabolic stress caused by intracellular bacteria triggers NLRP1B activation, but the mechanism by which this occurs was not elucidated. Here we demonstrate that TLR4 signaling in metabolically stressed macrophages promotes the formation of a TRIF/RIPK1/caspase-8 complex. Caspase-8 activity, induced downstream of this TLR4 pathway or through a distinct TNF receptor pathway, causes cleavage and activation of NLRP1B, which facilitates the maturation of both pro-caspase-1 and pro-caspase-8. Thus, our findings indicate that caspase-8 and NLRP1B generate a positive feedback loop that amplifies cell death processes and promotes a pro-inflammatory response through caspase-1. The ability of NLRP1B to detect caspase-8 activity suggests that this pattern recognition receptor may play a role in the defense against a variety of pathogens that induce apoptosis.

Anti-inflammatory potential of aspergillus unguis SP51-EGY: TLR4-dependent effects & chemical diversity via Q-TOF LC-HRMS.

Inflammation serves as an intricate defense mechanism for tissue repair. However, overactivation of TLR4-mediated inflammation by lipopolysaccharide (LPS) can lead to detrimental outcomes such as sepsis, acute lung injury, and chronic inflammation, often associated with cancer and autoimmune diseases. This study delves into the anti-inflammatory properties of "Aspergillus unguis isolate SP51-EGY" on LPS-stimulated RAW 264.7 macrophages. Through real-time qPCR, we assessed the expression levels of pivotal inflammatory genes, including iNOS, COX-2, TNF-α, and IL-6. Remarkably, our fungal extracts significantly diminished NO production and showed noteworthy reductions in the mRNA expression levels of the aforementioned genes. Furthermore, while Nrf2 is typically associated with modulating inflammatory responses, our findings indicate that the anti-inflammatory effects of our extracts are not Nrf2-dependent. Moreover, the chemical diversity of the potent extract (B Sh F) was elucidated using Q-TOF LC-HRMS, identifying 54 compounds, some of which played vital roles in suppressing inflammation. Most notably, compounds like granisetron, fenofibrate, and umbelliprenin were found to downregulate TNF-α, IL-1ß, and IL-6 through the NF-κB signaling pathway. In conclusion, "Aspergillus unguis isolate SP51-EGY", isolated from the Red Sea, Egypt, has been unveiled as a promising TLR4 inhibitor with significant anti-inflammatory potentials, presenting novel insights for their potential therapeutic use in inflammation.

Sheep (Ovis aries) Milk Exosomal miRNAs Attenuate Dextran Sulfate Sodium-Induced Colitis in Mice via TLR4 and TRAF-1 Inhibition.

Mammalian milk exosomal miRNAs play an important role in maintaining intestinal immune homeostasis and protecting epithelial barrier function, but the specific miRNAs and whether miRNA-mediated mechanisms are responsible for these benefits remain a matter of investigation. This study isolated sheep milk-derived exosomes (sheep MDEs), identifying the enriched miRNAs in sheep MDEs, oar-miR-148a, and oar-let-7b as key components targeting TLR4 and TRAF1, which was validated by a dual-luciferase reporter assay. In dextran sulfate sodium-induced colitis mice, administration of sheep MDEs alleviated colitis symptoms, reduced colonic inflammation, and systemic oxidative stress, as well as significantly increased colonic oar-miR-148a and oar-let-7b while reducing toll-like receptor 4 (TLR4) and TNF-receptor-associated factor 1 (TRAF1) level. Further characterization in TNF-α-challenged Caco-2 cells showed that overexpression of these miRNAs suppressed the TLR4/TRAF1-IκBα-p65 pathway and reduced IL-6 and IL-12 production. These findings indicate that sheep MDEs exert gastrointestinal anti-inflammatory effects through the miRNA-mediated modulation of TLR4 and TRAF1, highlighting their potential in managing colitis.

Targeting Toll-like Receptor 4/Nuclear Factor-κB and Nrf2/Heme Oxygenase-1 Crosstalk via Trimetazidine Alleviates Lipopolysaccharide-Induced Depressive-like Behaviors in Mice.

Depression is a global psychiatric illness that imposes a substantial economic burden. Unfortunately, traditional antidepressants induce many side effects which limit patient compliance thus, exploring alternative therapies with fewer adverse effects became urgent. This study aimed to investigate the effect of trimetazidine (TMZ); a well-known anti-ischemic drug in lipopolysaccharide (LPS) mouse model of depression focusing on its ability to regulate toll-like receptor 4 (TLR4)/nuclear factor-κB (NF-κB) as well as nuclear factor erythroid 2 related factor 2 (Nrf2)/ heme oxygenase-1 (HO-1) signaling pathways. Male Swiss albino mice were injected with LPS (500 µg/kg, i.p) every other day alone or parallel with oral doses of either escitalopram (Esc) (10 mg/kg/day) or TMZ (20 mg/kg/day) for 14 days. Treatment with TMZ attenuated LPS-induced animals' despair with reduced immobility time inforced swimming test. TMZ also diminished LPS- induced neuro-inflammation via inhibition of TLR4/NF-κB pathway contrary to Nrf2/HO-1 cascade activation with consequent increase in reduced glutathione (GSH) and HO-1 levels whereas the pro-inflammatory cytokines; tumor necrosis factor-α (TNF-α) and interleukin (IL)-1ß were evidently reduced. Besides, TMZ replenished brain serotonin levels via serotonin transporter (SERT) inhibition. Thus, TMZ hindered LPS-induced neuro-inflammation, oxidative stress, serotonin deficiency besides its anti-apoptotic effect which was reflected by decreased caspase-3 level. Neuroprotective effects of TMZ were confirmed by the histological photomicrographs which showed prominent neuronal survival. Here we showed that TMZ is an affluent nominee for depression management via targeting TLR4/NF-κB and Nrf2/HO-1 pathways. Future research addressing TMZ-antidepressant activity in humans is mandatory to enroll it as a novel therapeutic strategy for depression.

S100A9 and HMGB1 orchestrate MDSC-mediated immunosuppression in melanoma through TLR4 signaling.

BACKGROUND: Immunotherapies for malignant melanoma are challenged by the resistance developed in a significant proportion of patients. Myeloid-derived suppressor cells (MDSC), with their ability to inhibit antitumor T-cell responses, are a major contributor to immunosuppression and resistance to immune checkpoint therapies in melanoma. Damage-associated molecular patterns S100A8, S100A9, and HMGB1, acting as toll like receptor 4 (TLR4) and receptor for advanced glycation endproducts (RAGE) ligands, are highly expressed in the tumor microenvironment and drive MDSC activation. However, the role of TLR4 and RAGE signaling in the acquisition of MDSC immunosuppressive properties remains to be better defined. Our study investigates how the signaling via TLR4 and RAGE as well as their ligands S100A9 and HMGB1, shape MDSC-mediated immunosuppression in melanoma. METHODS: MDSC were isolated from the peripheral blood of patients with advanced melanoma or generated in vitro from healthy donor-derived monocytes. Monocytes were treated with S100A9 or HMGB1 for 72 hours. The immunosuppressive capacity of treated monocytes was assessed in the inhibition of T-cell proliferation assay in the presence or absence of TLR4 and RAGE inhibitors. Plasma levels of S100A8/9 and HMGB1 were quantified by ELISA. Single-cell RNA sequencing (scRNA-seq) was performed on monocytes from patients with melanoma and healthy donors. RESULTS: We showed that exposure to S100A9 and HMGB1 converted healthy donor-derived monocytes into MDSC through TLR4 signaling. Our scRNA-seq data revealed in patient monocytes enriched inflammatory genes, including S100 and those involved in NF-κB and TLR4 signaling, and a reduced major histocompatibility complex II gene expression. Furthermore, elevated plasma S100A8/9 levels correlated with shorter progression-free survival in patients with melanoma. CONCLUSIONS: These findings highlight the critical role of TLR4 and, to a lesser extent, RAGE signaling in the conversion of monocytes into MDSC-like cells, underscore the potential of targeting S100A9 to prevent this conversion, and highlight the prognostic value of S100A8/9 as a plasma biomarker in melanoma.

Disulfiram attenuates cell and tissue damage and blood‒brain barrier dysfunction after intracranial haemorrhage by inhibiting the classical pyroptosis pathway.

No single treatment significantly reduces the mortality rate and improves neurological outcomes after intracerebral haemorrhage (ICH). New evidence suggests that pyroptosis-specific proteins are highly expressed in the perihaematomal tissues of patients with ICH and that the disulfiram (DSF) inhibits pyroptosis. An ICH model was established in C57BL/6 mice by intracranial injection of collagenase, after which DSF was used to treat the mice. Cell model of ICH was constructed, and DSF was used to treat the cells. HE, TUNEL, Nissl, FJC and IF staining were performed to evaluate the morphology of brain tissues; Western blotting and ELISA were performed to measure the protein expression of NOD-like receptor protein 3 (NLRP3)/Caspase-1/gasdermin D (GSDMD) classical pyroptosis pathway and Toll-likereceptor4 (TLR4)/nuclear factor-kappaB (NF-κB) inflammatory signaling pathway and blood‒brain barrier-associated factoes, and the wet/dry weight method was used to determine the brain water content. The expression of proteins related to the NLRP3/Caspase-1/GSDMD pathway and the TLR4/NF-κB pathway was upregulated in tissues surrounding the haematoma compared with that in control tissues; Moreover, the expression of the blood-brain barrier structural proteins occludin and zonula occludens-1 (ZO-1) was downregulated, and the expression of Aquaporin Protein-4 (AQP4) and matrix metalloprotein 9 (MMP-9) was upregulated. DSF significantly inhibited these changes, reduced the haematoma volume, decreased the brain water content, reduced neuronal death and degeneration and improved neurological function after ICH. ICH activated the classical pyroptosis pathway and TLR4/NF-κB inflammatory pathway, disruped the expression of blood-brain barrier structural proteins, and exacerbated brain injury and neurological dysfunction. DSF inhibited these changes and exerted the therapeutic effects on pathological changes and dysfunction caused by ICH.

Morphine induces inflammatory responses via both TLR4 and cGAS-STING signaling pathways.

BACKGROUND: Opioid activation of the microglia or macrophage Toll-like receptor 4 (TLR4) and associated inflammatory cytokine release are implicated in opioid-induced hyperalgesia and tolerance. The cyclic GMP-AMP synthase/stimulator of interferon genes (cGAS-STING) signaling pathway, activated by double-stranded DNA including mitochondrial DNA (mtDNA), has emerged as another key mediator of inflammatory responses. This study tested the hypothesis that morphine induces immune inflammatory responses in microglia and macrophages involving TLR4 and cGAS-STING pathway. METHODS: BV2 microglia and Raw 264.7 (Raw) macrophage cells were exposed to morphine with and without a STING inhibitor (C176) for 6 h or TLR 4 inhibitor (TAK242) for 24 h. Western blotting and RT-qPCR analyses assessed TLR4, cGAS, STING, nuclear factor-kappa B (NF-κB), and pro-inflammatory cytokine expression. Morphine-induced mitochondria dysfunction was quantified by reactive oxygen species (ROS) release using MitoSOX, mtDNA release by immunofluorescence, and RT-qPCR. Polarization of BV2 and Raw cells was assessed by inducible nitric oxide (iNOS) and CD86 expression. The role of mtDNA on morphine-related inflammation was investigated by mtDNA depletion of the cells with ethidium bromide (EtBr) or cell transfection of mtDNA extracted from morphine-treated cells. RESULTS: Morphine significantly increased the expression of TLR4, cGAS, STING, p65 NF-κB, and cytokines (IL-6 and TNF-α) in BV2 and Raw cells. Morphine-induced mitochondrial dysfunction by increased ROS and mtDNA release; the increased iNOS and CD86 evidenced inflammatory M1-like phenotype polarization. TLR4 and STING inhibitors reduced morphine-induced cytokine release in both cell types. The transfection of mtDNA activated inflammatory signaling proteins, cytokine release, and polarization. Conversely, mtDNA depletion led to the reversal of these effects. CONCLUSION: Morphine activates the cGAS-STING pathway in macrophage cell types. Inhibition of the STING pathway can be an additional method to overcome immune cell inflammation-related morphine tolerance and opioid-induced hyperalgesia.

Proanthocyanidins alleviate Henoch-Schönlein purpura by mitigating inflammation and oxidative stress through regulation of the TLR4/MyD88/NF-κB pathway.

OBJECTIVE: Investigate Proanthocyanidins (PCs) efficacy and mechanisms in treating Henoch-Schönlein purpura (HSP)-like rat models, focusing on inflammatory and oxidative stress (OS) responses. METHODS: An HSP-like rat model was established using ovalbumin (OVA) injection, leading to symptoms mimicking HSP. The study measured inflammatory markers (IL-4, IL-17, TNF-α), OS markers (MDA, SOD, CAT), and assessed the TLR4/MyD88/NF-κB signaling pathway's involvement via histopathological and immunofluorescence analyses. RESULTS: PCs treatment significantly improved HSP-like symptoms, reduced inflammatory cell infiltration, and decreased IgA deposition in renal mesangial areas. Serum analyses revealed that PCs effectively lowered IL-4, IL-17, TNF-α, and MDA levels while increasing SOD and CAT levels (p < 0.05). Crucially, PCs also downregulated TLR4, MyD88, and NF-κB expressions, highlighting the blockage of the TLR4-mediated signaling pathway as a key mechanism. CONCLUSION: PCs show promising therapeutic effects in HSP-like rats by mitigating inflammatory responses and oxidative damage, primarily through inhibiting the TLR4/MyD88/NF-κB pathway. These findings suggest PCs as a potential treatment avenue for HSP, warranting further investigation.

Ginsenoside Rh2 Alleviates LPS-Induced Inflammatory Responses by Binding to TLR4/MD-2 and Blocking TLR4 Dimerization.

Lipopolysaccharide (LPS) triggers a severe systemic inflammatory reaction in mammals, with the dimerization of TLR4/MD-2 upon LPS stimulation serving as the pivotal mechanism in the transmission of inflammatory signals. Ginsenoside Rh2 (G-Rh2), one of the active constituents of red ginseng, exerts potent anti-inflammatory activity. However, whether G-Rh2 can block the TLR4 dimerization to exert anti-inflammatory effects remains unclear. Here, we first investigated the non-cytotoxic concentration of G-Rh2 on RAW 264.7 cells, and detected the releases of pro-inflammatory cytokines in LPS-treated RAW 264.7 cells, and then uncovered the mechanisms involved in the anti-inflammatory activity of G-Rh2 through flow cytometry, fluorescent membrane localization, Western blotting, co-immunoprecipitation (Co-IP), molecular docking and surface plasmon resonance (SPR) analysis in LPS-stimulated macrophages. Our results show that G-Rh2 stimulation markedly inhibited the secretion of LPS-induced interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α) and nitric oxide (NO). Additionally, G-Rh2 blocked the binding of LPS with the membrane of RAW 264.7 cells through direct interaction with TLR4 and MD-2 proteins, leading to the disruption of the dimerization of TLR4 and MD-2, followed by suppression of the TLR4/NF-κB signaling pathway. Our results suggest that G-Rh2 acts as a new inhibitor of TLR4 dimerization and may serve as a promising therapeutic agent against inflammation.

Tropomodulin1 exacerbates inflammatory response in macrophages by negatively regulating LPS-induced TLR4 endocytosis.

The excessive inflammation caused by the prolonged activation of Toll-like receptor 4 (TLR4) and its downstream signaling pathways leads to sepsis. CD14-mediated endocytosis of TLR4 is the key step to control the amount of TLR4 on cell membrane and the activity of downstream pathways. The actin cytoskeleton is necessary for receptor-mediated endocytosis, but its role in TLR4 endocytosis remains elusive. Here we show that Tropomodulin 1 (Tmod1), an actin capping protein, inhibited lipopolysaccharide (LPS)-induced TLR4 endocytosis and intracellular trafficking in macrophages. Thus it resulted in increased surface TLR4 and the upregulation of myeloid differentiation factor 88 (MyD88)-dependent pathway and the downregulation of TIR domain-containing adaptor-inducing interferon-ß (TRIF)-dependent pathway, leading to the enhanced secretion of inflammatory cytokines, such as TNF-α and IL-6, and the reduced secretion of cytokines, such as IFN-ß. Macrophages deficient with Tmod1 relieved the inflammatory response in LPS-induced acute lung injury mouse model. Mechanistically, Tmod1 negatively regulated LPS-induced TLR4 endocytosis and inflammatory response through modulating the activity of CD14/Syk/PLCγ2/IP3/Ca2+ signaling pathway, the reorganization of actin cytoskeleton, and the membrane tension. Therefore, Tmod1 is a key regulator of inflammatory response and immune functions in macrophages and may be a potential target for the treatment of excessive inflammation and sepsis.

Moxibustion with seed-size moxa cones alleviates colonic injury in mice with ulcerative colitis by regulating TLR4/MyD88/NF-κB signaling pathway. / 麦粒灸调控TLR4/MyD88/NF-κBä¿¡å·é€šè·¯å‡è½»æºƒç–¡æ€§ç»“è‚ ç‚Žå°é¼ ç»“è‚ æŸä¼¤çš„æœºåˆ¶ç ”ç©¶.

OBJECTIVES: To observe the effect of moxibustion with seed-size moxa cones on the Toll-like receptor 4(TLR4)/myeloid differentiation factor 88(MyD88)/nuclear transcription factor-κB(NF-κB) signaling pathway in mice with ulcerative colitis(UC), so as to explore the therapeutic mechanism of moxibustion with seed-size moxa cones on colonic injury in UC. METHODS: Forty male C57BL/6 mice were randomly divided into blank group, model group, moxibustion group, and western medicine group, with 10 mice in each group. The UC mouse model was established by 3% DSS solution by free drinking for 7 consecutive days. Mice in the moxibustion group were treated with seed-size moxa cones at "Zhongwan"(CV12), "Tianshu"(ST25) and "Shangjuxu"(ST37), 3 moxa cones per point, with each cone applied for approximately 30 s, while mice in the western medicine group were orally administered with 300 mg/kg mesalazine solution, which were both conducted once a day for 7 consecutive days. The general condition of mice was observed every 2 days, and the disease activity index (DAI) score was calculated. HE staining was used to observe the morphology of colonic tissue in mice. ELISA was used to detect the serum interleukin(IL)-1ß, tumor necrosis factor(TNF)-α, IL-6, and IL-8 contents. Immunohistochemistry was used to detect the positive expression of TLR4 and MyD88 in colonic tissue of mice. Real-time fluorescence quantitative PCR was used to detect the expression levels of TLR4, MyD88, and NF-κB p65 mRNAs in colonic tissue. RESULTS: Compared with the blank group, varying degrees of soft or watery stools were observed, colon length and body weight were decreased(P<0.01) in mice of the model group, while DAI score, colon weight index, mucosal damage score, colonic pathological score, serum IL-1ß, TNF-α, IL-6, and IL-8 contents, positive expressions of TLR4 and MyD88, and TLR4, MyD88, and NF-κB p65 mRNA expressions in colonic tissue were increased(P<0.01). Compared with the model group, improved fecal characteristics were observed, colon length and body weight were increased(P<0.01) in mice of the moxibustion group and western medicine group, while DAI scores, colon weight indexes, mucosal damage scores, colonic pathological score, serum contents of IL-1ß, TNF-α, IL-6, and IL-8, positive expressions of TLR4 and MyD88, and TLR4, MyD88, and NF-κB p65 mRNA expressions in colonic tissue were decreased(P<0.01, P<0.05). There was no significant difference in the above indicators between the moxibustion group and the western medicine group. CONCLUSIONS: Moxibustion with seed-size moxa cones may alleviate colonic injury in UC mice by regulating the TLR4/MyD88/NF-κB signaling pathway and reducing the release of inflammatory factors.

Multiomics integrated analysis and experimental validation identify TLR4 and ALOX5 as oxidative stress-related biomarkers in intracranial aneurysms.

BACKGROUND: Intracranial aneurysm (IA) is a severe cerebrovascular disease, and effective gene therapy and drug interventions for its treatment are still lacking. Oxidative stress (OS) is closely associated with the IA, but the key regulatory genes involved are still unclear. Through multiomics analysis and experimental validation, we identified two diagnostic markers for IA associated with OS. METHODS: In this study, we first analyzed the IA dataset GSE75436 and conducted a joint analysis of oxidative stress-related genes (ORGs). Differential analysis, functional enrichment analysis, immune infiltration, WGCNA, PPI, LASSO, and other methods were used to identify IA diagnostic markers related to OS. Next, the functions of TLR4 and ALOX5 expression in IA and their potential targeted therapeutic drugs were analyzed. We also performed single-cell sequencing of patient IA and control (superficial temporal artery, STA) tissues. 23,342 cells were captured from 2 IA and 3 STA samples obtained from our center. Cell clustering and annotation were conducted using R software to observe the distribution of TLR4 and ALOX5 expression in IAs. Finally, the expression of TLR4 and ALOX5 were validated in IA patients and in an elastase-induced mouse IA model using experiments such as WB and immunofluorescence. RESULTS: Through bioinformatics analysis, we identified 16 key ORGs associated with IA pathogenesis. Further screening revealed that ALOX5 and TLR4 were highly expressed to activate a series of inflammatory responses and reduce the production of myocytes. Methotrexate (MTX) may be a potential targeted drug. Single-cell analysis revealed a notable increase in immune cells in the IA group, with ALOX5 and TLR4 primarily localized to monocytes/macrophages. Validation through patient samples and mouse models confirmed high expression of ALOX5 and TLR4 in IAs. CONCLUSIONS: Bioinformatics analysis indicated that ALOX5 and TLR4 are the most significant ORGs associated with the pathogenesis of IA. Single-cell sequencing and experiments revealed that the high expression of ALOX5 and TLR4 are closely related to IA. These two genes are promising new targets for IA therapy.

Zafirlukast ameliorates lipopolysaccharide and bleomycin-induced lung inflammation in mice.

Acute lung injury (ALI) is the result of damage to the capillary endothelia and the alveolar epithelial cell caused by various direct and indirect factors, leading to significant pulmonary interstitial and alveolar edema and acute hypoxic respiratory insufficiency. A subset of ALI cases progresses to irreversible pulmonary fibrosis, a condition with fatal implications. Zafirlukast is a leukotriene receptor antagonist licensed for asthma prevention and long-term treatment. This study demonstrated a significant improvement in lung tissue pathology and a reduction in inflammatory cell infiltration in models of lipopolysaccharide (LPS)-induced ALI and bleomycin (BLM)-induced lung inflammation following zafirlukast administration, both in vivo and in vitro. Moreover, zafirlukast was found to suppress the inflammatory response of alveolar epithelial cells in vitro and lung inflammation in vivo by reducing the activation of the TLR4/NF-κB/NLRP3 inflammasome pathway. In conclusion, zafirlukast relieved lung injury and the infiltration of inflammatory cells in the lung by regulating the TLR4/NF-κB/NLRP3 pathway.

[Linarin inhibits microglia activation-mediated neuroinflammation and neuronal apoptosis in mouse spinal cord injury by inhibiting the TLR4/NF-κB pathway].

OBJECTIVE: To investigate the mechanism underlying the neuroprotective effect of linarin (LIN) against microglia activation-mediated inflammation and neuronal apoptosis following spinal cord injury (SCI). METHODS: Fifty C57BL/6J mice (8- 10 weeks old) were randomized to receive sham operation, SCI and linarin treatment at 12.5, 25, and 50 mg/kg following SCI (n=10). Locomotor function recovery of the SCI mice was assessed using the Basso Mouse Scale, inclined plane test, and footprint analysis, and spinal cord tissue damage and myelination were evaluated using HE and LFB staining. Nissl staining, immunofluorescence assay and Western blotting were used to observe surviving anterior horn motor neurons in injured spinal cord tissue. In cultured BV2 cells, the effects of linarin against lipopolysaccharide (LPS)­induced microglia activation, inflammatory factor release and signaling pathway changes were assessed with immunofluorescence staining, Western blotting, RT-qPCR, and ELISA. In a BV2 and HT22 cell co-culture system, Western blotting was performed to examine the effect of linarin against HT22 cell apoptosis mediated by LPS-induced microglia activation. RESULTS: Linarin treatment significantly improved locomotor function (P < 0.05), reduced spinal cord damage area, increased spinal cord myelination, and increased the number of motor neurons in the anterior horn of the SCI mice (P < 0.05). In both SCI mice and cultured BV2 cells, linarin effectively inhibited glial cell activation and suppressed the release of iNOS, COX-2, TNF-α, IL-6, and IL-1ß, resulting also in reduced neuronal apoptosis in SCI mice (P < 0.05). Western blotting suggested that linarin-induced microglial activation inhibition was mediated by inhibition of the TLR4/NF- κB signaling pathway. In the cell co-culture experiments, linarin treatment significantly decreased inflammation-mediated apoptosis of HT22 cells (P < 0.05). CONCLUSION: The neuroprotective effect of linarin is medicated by inhibition of microglia activation via suppressing the TLR4/NF­κB signaling pathway, which mitigates neural inflammation and reduce neuronal apoptosis to enhance motor function of the SCI mice.

Programming Bordetella pertussis lipid A to promote adjuvanticity.

BACKGROUND: Bordetella pertussis is the causative agent of whooping cough or pertussis. Although both acellular (aP) and whole-cell pertussis (wP) vaccines protect against disease, the wP vaccine, which is highly reactogenic, is better at preventing colonization and transmission. Reactogenicity is mainly attributed to the lipid A moiety of B. pertussis lipooligosaccharide (LOS). Within LOS, lipid A acts as a hydrophobic anchor, engaging with TLR4-MD2 on host immune cells to initiate both MyD88-dependent and TRIF-dependent pathways, thereby influencing adaptive immune responses. Lipid A variants, such as monophosphoryl lipid A (MPLA) can also act as adjuvants. Adjuvants may overcome the shortcomings of aP vaccines. RESULTS: This work used lipid A modifying enzymes from other bacteria to produce an MPLA-like adjuvant strain in B. pertussis. We created B. pertussis strains with distinct lipid A modifications, which were validated using MALDI-TOF. We engineered a hexa-acylated monophosphorylated lipid A that markedly decreased human TLR4 activation and activated the TRIF pathway. The modified lipooligosaccharide (LOS) promoted IRF3 phosphorylation and type I interferon production, similar to MPLA responses. We generated three other variants with increased adjuvanticity properties and reduced endotoxicity. Pyrogenicity studies using the Monocyte Activation Test (MAT) revealed that these four lipid A variants significantly decreased the IL-6, a marker for fever, response in peripheral blood mononuclear cells (PBMCs). CONCLUSION: These findings pave the way for developing wP vaccines that are possibly less reactogenic and designing adaptable adjuvants for current vaccine formulations, advancing more effective immunization strategies against pertussis.

IL36G-producing neutrophil-like monocytes promote cachexia in cancer.

Most patients with advanced cancer develop cachexia, a multifactorial syndrome characterized by progressive skeletal muscle wasting. Despite its catastrophic impact on survival, the critical mediators responsible for cancer cachexia development remain poorly defined. Here, we show that a distinct subset of neutrophil-like monocytes, which we term cachexia-inducible monocytes (CiMs), emerges in the advanced cancer milieu and promotes skeletal muscle loss. Unbiased transcriptome analysis reveals that interleukin 36 gamma (IL36G)-producing CD38+ CiMs are induced in chronic monocytic blood cancer characterized by prominent cachexia. Notably, the emergence of CiMs and the activation of CiM-related gene signatures in monocytes are confirmed in various advanced solid cancers. Stimuli of toll-like receptor 4 signaling are responsible for the induction of CiMs. Genetic inhibition of IL36G-mediated signaling attenuates skeletal muscle loss and rescues cachexia phenotypes in advanced cancer models. These findings indicate that the IL36G-producing subset of neutrophil-like monocytes could be a potential therapeutic target in cancer cachexia.

Structural identification, rheological properties and immunological receptor of a complex galacturonoglucan from fruits of Schisandra chinensis (Turcz.) Baill.

A complex heteropolysaccharide SCP-2 named schisanan B (Mw = 1.005 × 105 g/mol) was obtained from water extracts of Schisandra chinensis fruits, and its planar structure was finally deduced as a galacturonoglucan by a combination of monosaccharide compositions, methylation analysis, partial acid hydrolysis, enzymatic hydrolysis and 1D/2D-nuclear magnetic resonance spectroscopy. The conformation of SCP-2 exhibited a globular shape with branching in ammonium formate aqueous solutions. The rheological properties of SCP-2 were investigated on concentrations, temperature, pH and salts. The in vitro immunomodulatory activity assay demonstrated that SCP-2 significantly enhanced the production of nitric oxide (NO) and stimulated the secretion of tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) in macrophages. Through a combination of high-resolution live-cell imaging, surface plasmon resonance, and molecular docking techniques, SCP-2 exhibited a strong binding affinity with the Toll-like receptor 4 (TLR4). Moreover, western blot analysis revealed that SCP-2 effectively induced downstream signaling proteins associated with TLR4 activation, thereby promoting macrophage activation. The evidence strongly indicates that TLR4 functions as a membrane protein target in the activation of macrophages and immune regulation induced by SCP-2.